Westcott.Project Summary/Abstract Enterotoxigenic E. coli (ETEC), Shigella flexneri and Campylobacter jejuni are the predominant bacterial causes of morbidity and mortality from gastrointestinal (GI) disease in the developing world. Episodes of acute diarrhea caused by these agents have been linked to developmental defects and chronic GI syndromes. Vaccines that are safe for use in young children as well as adult travelers and deployed military are needed to prevent infection and post-infection sequellae caused by these organisms. Formalin inactivation is the current standard for generating killed, whole cell bacterial vaccines. This approach does not require genetic manipulations or extensive knowledge of antigens that induce protective immune responses. However, formalin crosslinks proteins, which has the potential to destroy antigenic epitopes that induce protective immune responses in the context of live bacterial infection. We propose to test the feasibility of an alternative inactivation method to generate safe, whole cell vaccines for enteric bacteria. Psoralen is a photoactivatable drug that reversibly intercalates into nucleic acids. Following irradiation with long wavelength UV light (P+UVA), covalent inter-strand crosslinks form at pyrimidine residues, preventing genome replication. Since P+UVA inactivates bacteria by crosslinking nucleic acids rather than proteins, we hypothesize that protein antigens will be preserved in a native form, generating vaccines with superior immunogenicity. We have successfully inactivated ETEC using P+UVA. With this proposal we will extend the ETEC work by comparing P+UVA to formalin-inactivated ETEC with respect to antigen preservation and immunogenicity using established in vitro assays and murine models. A direct comparative analysis of the 2 inactivation methods is needed to assess the value and feasibility of the P+UVA approach as an agile platform for enteric bacterial vaccines, and more generally for vaccines to prevent antibiotic-resistant bacterial infections that impact patient care in hospital settings. . 1